Known models for simulating blood vessels and the organotypic culture surrounding them have a large number of drawbacks, in particular with respect to the form of the vessels, the density of different capillaries in the organotypic culture or the porosity of channels which ensure the active supply to the organotypic culture, including gradient formation. However, the complexity of the vascular system requires close to in vivo representation of the physiological conditions, in particular the radii of curvature of the blood vessels, interactions with the extracellular matrix and cells or shear forces.
With known methods for producing microchannel structures, in particular lithography (preferably X-ray, UV or laser radiation), mechanical microstructuring, laser structuring, soft lithography, microreplication methods such as injection moulding or hot stamping, or rapid prototyping methods, microfluidic channels having a rectangular cross-section are usually obtained. Cells which are cultured in channels of this type grow on a planar surface, but this does not correspond to the in vivo conditions [1]. With many microtechnical methods, channels having a round cross-section can be produced in a complex manner, but typically these are not porous [2-6]. However, pores are necessary for supplying cells both from the apical side and from the basolateral side and for examining transendothelial transport processes.
It is known that a three-dimensional tissue which is located in the immediate vicinity of a blood vessel secretes factors which can increase transendothelial transport. This is particularly relevant in the case of tumour tissues, since they secrete and/or recruit proteases which promote short-time breakdown of the endothelial connectivity by proteolysis of what are known as tight junctions, and thus make the blood vessel permeable for active agent transport.
It has been shown that the morphology of the endothelial cells and the correct formation of tight junctions between the endothelial cells in straight rectangular channels differ significantly from those in rounded channels, not only under static conditions but also under fluidic conditions. In addition, flow profiles in rectangular microchannels differ from flow profiles in microchannels having a circular cross-section, whereby the differentiation of the endothelial cells which can be cultured in these channels also varies greatly [2, 7]. Anomalies in the formation and the size of the plasma-rich layer owing to red blood cells were also observed in rectangular fluidic microchannels [8]. Morphology studies on endothelial cells in microchannels having rectangular and circular cross-sections showed that there are particularly great differences in the correct formation of the actin cytoskeleton and the focal adhesion points [9]. The morphology of the endothelial cells and the correct formation of tight junctions between the endothelial cells differ significantly not only under fluidic conditions but also under static conditions.
A three-dimensional structure corresponding to the natural environment represents the in vivo situation of the cells better than a planar surface [10-12]. Since the vascular system is a complex cellular system, it is very important for an in vitro system to provide as far as possible the physiological environment, in particular curvature of the vessel structures, composition of the extracellular matrix, fluidics, shear force ratio, density of supply to the three-dimensional tissue with channels at small intervals, branching of the channels [12, 13]. Most microfluidic channel structure systems are produced from polydimethylsiloxanes (PDMS) by soft lithography. In [2], a microfluidic channel system made of PDMS and having a circular cross-section is presented, which system makes it possible to imitate and examine cardiovascular flow conditions in endothelial cells. However, this system is not suitable for examining transendothelial transport of active agents in tissue, since these are closed channels, the outer shell of which consists mainly of a collagen matrix.
At present, there are various artificial models of round perfused channels based on hollow fibres as artificial blood vessels [14]. However, in all hollow fibre systems, gradients can occur in the longitudinal and the radial direction of the fibres, since the culture medium must take a particular route from the capillary inlet to the capillary outlet, along which route the cells deplete the nutrients in the culture medium. The provision of nutrition and blood flow through blood vessels is a significant unsolved problem in the establishment of an organotypic model. Even in the case of small tissue volumes it is important to implement a vascular system or a corresponding equivalent, since for distances of more than approximately 100-300 μm to the nearest blood capillary the diffusion is no longer sufficient for nutrition. There is therefore a need recognized in the present invention for models in which supplying blood vessels can be cultured in combination with any desired tissue and which also meet the requirements of a (micro)vascular system. The provision of a physiologically correct, three-dimensional arrangement is thus required, and this has not yet been achieved with conventional hollow fibre systems, since hollow fibres allow neither branching nor frequent change of the cross-sections.
Systems as described in [19] are based on the repopulation of acellularised porcine small intestine segments, the vascular system of which is repopulated with endothelial cells. Various three-dimensional tissues can be cultured on the outer surface of the blood vessels and can be supplied via the repopulated blood vessels. However, the system needs further optimisation with regard to the supply to the tissue. The variability in the explantation and acellularisation leads to different qualities of the individual matrices. Moreover, this model cannot be used as a single-use product for testing in the high-throughput method.
In [15], a very simple strategy for the use of thermoformed, thin-walled and porous channels for supplying thick three-dimensional tissues is proposed. However, this document does not consider active perfusion of the channels for supplying the cells. Cells in the hydrogel are supplied only by diffusion from the channels, which are not actively perfused.